RCEES OpenIR  > 环境化学与生态毒理学国家重点实验室
成团泛菌介导的贵金属还原机制与应用研究
Alternative TitleMechanism and application of noble metal reduction mediated by Pantoea sp. IMH
刘文婧
Subtype博士
Thesis Advisor景传勇
2018-12
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
Keyword微生物,贵金属,生物合成纳米颗粒,拉曼成像,催化转化 microorganism, Noble Metal, Biological Synthesized Nanoparticles, Raman Imaging, Catalytic Reductive Transformation
Abstract

      微生物在贵金属离子的生物地球化学循环过程中起着至关重要的作用。一定浓度的贵金属离子对微生物细胞具有毒性效应,为了抵御其毒性,微生物细胞发展进化了多种金属抗性系统以保护胞内组分。微生物能够在酶催化作用下对贵金属进行氧化还原反应,从而改变离子的氧化还原状态,如形成零价金属颗粒沉淀或生成有机金属小分子化合物。现有研究已发现多种细菌能够在胞外和胞内合成金属纳米颗粒,但微生物对贵金属离子的抗性及还原机制尚不清楚,有待进一步研究。基于微生物金属抗性合成生物相容、安全清洁、经济环保的纳米颗粒具有巨大的研究价值及应用潜力,因此探索贵金属纳米颗粒的绿色合成及应用成为了亟待关注的问题。成团泛菌Pantoea sp. IMH是从高砷环境分离的菌株,并具有多种金属抗性基因簇。本文以Pantoea sp. IMH为代表菌株,旨在:1)探索Pantoea sp. IMH对金属银、钯和硒离子的抗性还原机制;2)研究Pantoea sp. IMH对金离子的还原过程与机制;3)基于Pantoea sp. IMH胞内还原银构建单细胞拉曼成像研究;4)并将此拉曼成像结合数据处理的方法应用拓展到生物组织判别分析;5)同时基于Pantoea sp. IMH胞内还原钯设计制备钯基复合材料,并对探索其催化应用。
      第一,本研究通过对银、钯和硒离子胁迫后的Pantoea sp. IMH细胞进行转录组分析,可知在贵金属离子胁迫下细胞差异表达的基因主要功能包括金属结合、转运和催化活性。且在将银、钯和硒离子胁迫过程中,分别形成了银、钯和硒纳米颗粒。经转录组分析葡萄糖参与了细胞还原Ag(I)过程;周质蛋白细胞色素CpxP参与了Pd(II)还原;而细胞对Se(IV)的抗性还原主要是通过草酸转运基因oxlT和As抗性基因簇。本研究系统地分析了单一菌株对多种金属离子的抗性和还原机制。
      第二,本研究探索了Pantoea sp. IMH对Au(III)的还原过程和机制。在金离子胁迫下,成团泛菌IMH生成具有核壳结构的金@生物膜纳米颗粒,且生物膜是由膜蛋白、脂蛋白和磷脂组成的。过程实验表明,Au NPs首先出现在胞外空间,最后出现在细胞质空间中。在Au(III)生物还原过程中,Au(I)是从Au(III)还原到Au(0)的中间体。Pantoea sp. IMH能够通过多种机制还原Au(III),包括通过胞外EPS的缩醛基还原和胞内fucO编码的还原酶还原为Au NPs。所生成的金纳米颗粒可用于环境污染物的选择性吸附和表面增强拉曼检测分析。这些结果加深了对生物合成纳米金的结构和还原机制的理解。
      第三,本研究在Pantoea sp. IMH原位还原Ag(I)合成纳米银的基础上,实现了单细胞拉曼成像。细胞还原合成的纳米银均匀分散在细胞内部。基于纳米银的表面增强拉曼(SERS)活性,构建了单细胞的二维和三维拉曼成像。通过将多变量数据分析方法应用于拉曼成像分析可知,多元曲线分辨分析(MCR)模型能够有效提取生物信息数据,验证了葡萄糖参与了Ag(I)在IMH细胞内的还原,同时细胞色素C与NADH依赖的还原酶也参与其中。本研究基于细胞原位还原银离子合成纳米银,构建了单细胞的化学成像方法,同时探索了细胞对银离子的还原机制理解。
       第四,在细胞拉曼成像与数据处理方法结合的基础上,本研究将此分析方法拓展到应用于组织细胞的判别分析。通过将细胞质与细胞核的差减光谱作为组织细胞的特征光谱,结合偏最小二乘分析(Partial least squares-discriminant analysis,PLS-DA)模型,构建了亚细胞水平的组织细胞的拉曼判别方法。将此方法应用于实际结肠细胞研究,实现了对癌细胞的判别分析。此方法不仅具有高稳定性,且对于直肠组织也具有优良的判别效果。上述结果表明该亚细胞拉曼分析方法在临床癌细胞判别分析中具备应用潜力。
       最后,本研究基于Pantoea sp. IMH细胞还原Pd(II)合成纳米钯,实现了纳米钯负载的氮掺杂碳材料(Pd@NC)的绿色合成。多种表征技术证明粒径为7 nm的纳米钯均匀分布在氮掺杂的碳层上。Pd@NC对硝基苯化合物具有优异的催化降解性能。X射线近边吸收结构分析和密度泛函理论计算结合分析,可知在氮原子掺杂的作用下,Pd@NC结构中形成Pd 4d和C 2p的杂化轨道,最终使得d电子空穴数增加,所以Pd@NC材料具有优异的催化活性。本研究合成的Pd@NC复合材料是一种具有环境应用潜力的催化材料,为金属-碳材料催化剂的制备提出了绿色合成方法。

Other Abstract

       Microorganisms play an important role in the biogeochemical cycle of noble metal in the environment. Metals at high concentration are toxic to microorganisms. Bacteria have adapted to metals through a variety of chromosomal, transposon, and plasmid-mediated resistance systems. Through enzymatic detoxification system, cells could alter the redox state of the metal, create metal crystal precipitates or generate organometallic small-molecule compounds. In recent research, bacteria have been explored for metal nanoparticle synthesis extracellularly and intracellularly.  Nevertheless, a grand challenge remains in deciphering the molecular mechanism regulating the metal ion tolerance and reduction in bacteria. Meanwhile, microorganisms have been shown to be important nanofactories that hold immense potential as ecofriendly and cost-effective tools. Metal nanoparticles have been successfully applied in the environmental pollutants remediation and characterization analysis areas. Thus, the application of nanoparticles synthesized by biological method needs to be explored. Herein, by chosing IMH as a representative strain, our purpose is: 1) to investigate the molecular mechanism of resistance to and reduction of Ag, Pd, and Se in bacteria; 2) to propose SERS spectral imaging of the single cell by in situ formed Ag nanoparticles to explore the biosynthesis mechanisms; 3) to extend the application in the human cancer diagnosis; 4) to achieve a green synthesis method for Pd NPs@N-doped carbon material.
       Firstly, incubation of Pantoea sp. IMH with Ag(I), Pd(II), and Se(IV) resulted in the formation of AgNPs, PdNPs, and SeNPs. Multiple complementary techniques including transmission electron microscopy (TEM), energy-dispersive spectrometry (EDS), and X-ray photoelectron spectroscopy (XPS) were used to characterize the biogenic nanoparticles. Transcriptome analysis shows that most of the genes differentially expressed under the stress of Ag(I), Pd(II), and Se(IV) were related to metal binding, transport, catalytic activity, and metabolic pathways. Meanwhile, the expression of gluconate metabolism in the strain IMH was specifically induced by Ag(I); the periplasmic protein cytochrome CpxP was involved in the reduction of Pd(II); and oxlT and ars gene clusters were expressed specifically in the Se(IV)-treated cells. Our study systematically analyzed the mechanisms of reduction of and resistance to multiple metals in a single strain, and shed new light on the metal resistance system.
       Secondly, our study describe the process and mechanism of Au@biolayer core-shell NPs biosynthesized by Pantoea sp. IMH. Our multiple complementary characterization results provided the first direct evidence of a biolayer on biogenic Au NPs. This 3-nm thick biolayer was comprised of membrane proteins, lipoproteins, and phospholipids, as determined by liquid chromatography-mass spectrometry/mass spectrometry. The Au NPs occurred first in the culture medium, then on the cell wall, and finally in the cytoplasmic space. Au(I) was detected as an intermediate in the bioreduction process by X-ray absorption near-edge structure spectroscopy. Pantoea sp. IMH utilizes several Au(III) reducing mechanisms, including reduction of Au(III) by acetal groups of exopolysaccharides in aqueous extracellular polymeric substances (EPS) outside the cell, reduction of Au(III) on cell walls by the EPS adhering to cell surfaces, and protein/enzymatic reduction in the cytoplasm involving fucO, glutathione, and metal resistance proteins. Our results shed new light on the structure and dynamic process of biosynthesizing Au NPs.
       Thirdly, our study proposed SERS spectral imaging of the single cell by in situ formed Ag nanoparticles to explore the biosynthesis mechanisms. HAADF-STEM were used to characterize the even-distributed biogenic Ag nanoparticles in cells. Raman mapping (2D/3D) was emplored in the single cell based on the intracellular Ag NPs. Moreover, the MCR model has been successfully applied in Raman mapping analysis, providing the analysis of biosynthesis mechanisms. The work described herein enhanced our understanding of intracellular AgNP biosynthesis by bacteria, providing chemical information to complement electron microscopy.
      Fourthly, as the application extension, we proposed the subcellular subtraction spectra of the nucleus and cytoplasm (Nuc.-Cyt.) as a straightforward Raman signature for colon tumor diagnosis. PLS-DA analysis for the subtraction spectra resulted in the excellent diagnostic performance for colon and rectal tissues as well as good stability. The results of this work suggest that our subcellular Raman analysis approach might be a powerful tool in improving cancer diagnosis during clinical examination.
       Finally, Pd@NC was synthesized via Pd(II) reduction by Pantoea sp. IMH followed by calcination. The green-synthesized Pd@NC exhibited exceptional catalytic activity for the reductive transformation of nitroaromatic compounds due to the unique structure of Pd@NC. This structure resulted not only in the uniform dispersion of Pd NPs, but also in the special electronic structure. The d-hole counts increase due to Pd 4d-C 2p hybridization in the presence of nitrogen doping, which correlates to the pronounced catalytic activity. This Pd@NC composite is a promising catalytic material, and the green synthesis route opens an avenue to the recovery of Pd and preparation of multifunctional metal-carbon catalysts for the degradation of organic pollutants.

Pages158
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42255
Collection环境化学与生态毒理学国家重点实验室
Recommended Citation
GB/T 7714
刘文婧. 成团泛菌介导的贵金属还原机制与应用研究[D]. 北京. 中国科学院生态环境研究中心,2018.
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